Visualizing the structures of biological molecular machines is crucial for a mechanistic understanding of cellular function. Cryo-EM is an emergent structural biology technique that is ideally suited to study large macromolecular assemblies, even if they are only available in minute amounts, or if multiple states co-exist in th sample. The cryo-EM field has just entered a new era with the use of high-end electron microscopes coupled to new direct electron detection devices (DDDs) that has led to high resolution structural information, in some cases from only one or a few imaging sessions. As technical barriers open up and the field matures, the impact of new cryo-EM studies will more and more be defined by the output of biological insight. Such will be measured by the relevance of the biological system under study and by whether the experimental strategy is able to provide answers to key functional questions. This scenario requires the synergistic integration of cryo-EM and biochemical expertise in the context of studies that address fundamental biological questions. This program project brings together such set of complementary expertise and provides a mechanism to integrate them seamlessly to tackle biological studies of great medical significance, providing a supportive and coherent environment that promotes interactions among the different projects and advances each of them individually, as well as the cryo-EM field as a whole. This Program Project also provides a mechanism whereby the large costs of operating high-end electron microscopes, associated equipment and computational capabilities are effectively shared among a number of research projects that rely on such state-of-the-art instrumentation. As cryo-EM and image reconstruction are still developing rapidly, optimal use of this methodology requires rigorous retraining and is best facilitated by specialized support personnel and intensive discussion among members of a collaborative group as proposed in this program project. Our ultimate goal is to maintain and enhance Berkeley's contributions to the structural biology field. We will support and employ a physical and intellectual infrastructure that allows us to carry out state-of-the-art cryo-EM studies of essential macromolecular complexes and push the capabilities of the cryo-EM technique. This proposal capitalizes on (1) the tradition of innovation and risk-taking within our PPG and years of leadership and expertise in the field of biological electron microscopy and structural biology, (2) the implementation at Berkeley of recent technical breakthroughs in the cryo-EM field (e.g. DDDs, automation, Bayesian methods of image processing), (3) unique biochemical expertise in a large number of biological systems of outstanding biomedical relevance, and (4) the unique local computer resources at the National Energy Research Scientific Computing Center (NERSC), which is hosted at LBNL.